The high degree of heterogeneity reported for prolactin-secreting cells and the variants of prolactin released necessitate analysis of mammotrope function at the single cell level. Such studies are now possible due to the development of reverse hemolytic plaque assays. These assays are based on antibody-directed, complement-mediated lysis of red blood cells in the vicinity of hormone secretors and they enable the microscopic visualization of hormone release from single cells in culture. The primary objectives of the proposed research are: 1) to evaluate the relative importance of dopamine and direct auto-feedback suppression by prolactin itself to the inhibitory control of prolactin release; 2) to establish a truly quantitative relationship between plaque size and the amount of hormone released from individual pituitary cells and then assess the absolute degree to which mammotropes differ from one another in their capacity to secrete hormone under basal and regulated conditions; and 3) to complete development of an assay capable of evaluating the bioactivity of prolactin released from a single cell and then use this system to study the microheterogeneity of secreted prolactin. These goals will be accomplished by employing two types of reverse hemolytic plaque assays. A standard version will be used to measure the amount of prolactin released from individual mammotropes. On the other hand, a novel "plaque-bioassay" based on casein release from cultured mammary cells will be employed to assess the biopotency of secreted prolactin. These studies will further our understanding of the functional relevance of heterogeneity at the cellular and molecular levels. Moreover, this knowledge can eventually be used for identifying the causes of, and developing treatments for, pathologies of prolactin secretion in humans.